17β-雌二醇对海马神经干细胞增殖和分化的影响及其机制  

Influence of 17β-estradiol in proliferation and differentiation of hippocampal neural stem cells and its mechanism

作　　者：杨英 赵亮 由涌 许倩[4] 杨振军 YANG Ying;ZHAO Liang;YOU Yong;XU Qian;YANG Zhenjun(Department of Human Anatomy,School of Basic Medical Sciences,Chengde Medical University,Chengde 067000,China;Department of Pharmacology,School of Basic Medical Sciences,Chengde Medical University,Chengde 067000,China;Department of Immunology,School of Basic Medical Sciences,Chengde Medical University,Chengde 067000,China;Institute of Basic Medicine,Chengde Medical University,Chengde 067000,China)

机构地区：[1]承德医学院基础医学院人体解剖教研室,河北承德067000 [2]承德医学院基础医学院药理学教研室,河北承德067000 [3]承德医学院基础医学院免疫学教研室,河北承德067000 [4]承德医学院基础医学研究所,河北承德067000

出　　处：《吉林大学学报(医学版)》2025年第2期317-324,共8页Journal of Jilin University:Medicine Edition

基　　金：国家自然科学基金项目(81700310);承德医学院高层次人才科研启动基金项目(202108)。

摘　　要：目的:观察17β-雌二醇(17β-E2)对原代培养海马神经干细胞(NSCs)增殖和分化能力的影响,并阐明其可能的作用机制。方法:提取新生24 h内SD大鼠海马组织NSCs,分为对照组和17β-E2组。采用免疫荧光法检测2组NSCs中巢蛋白(Nestin)和5-乙炔基-2′-脱氧尿苷(EdU)的表达情况,计算NSCs增殖率。Western blotting法检测NSCs中Nestin蛋白表达水平,流式细胞术检测不同细胞周期NSCs百分率。采用免疫荧光法检测NSCs分化后神经元标志物βⅢ-微管蛋白(βⅢ-tubulin)和星形胶质细胞标志物胶质纤维酸性蛋白(GFAP)的表达情况,计算神经元与星形胶质细胞的相对比值。Western blotting法检测NSCs分化后βⅢ-tubulin、GFAP以及磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、糖原合成激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)和β-连环蛋白(β-catenin)等PI3K/Akt/GSK-3β通路相关蛋白的表达水平。结果:免疫荧光法,2组NSCs中Nestin均阳性表达,与对照组比较,17β-E2组NSCs增殖率明显升高(P<0.01);Western blotting法,与对照组比较,17β-E2组NSCs中Nestin蛋白表达水平升高(P<0.05)。流式细胞术,与对照组比较,17β-E2组S期NSCs百分率明显升高(P<0.01)。免疫荧光法,与对照组比较,17β-E2组神经元与星形胶质细胞的相对比值明显升高(P<0.05)。Western blotting法,与对照组比较,17β-E2组NSCs分化后βⅢ-tubulin蛋白表达水平升高(P<0.05),GFAP蛋白表达水平降低(P<0.01),Akt、p-Akt、β-catenin和p-GSK-3β蛋白表达水平升高(P<0.05或P<0.01),GSK-3β蛋白表达水平降低(P<0.05)。结论:17β-E2可以促进NSCs增殖并促使其向神经元方向分化,其作用机制可能与PI3K/Akt/GSK-3β信号通路有关。Objective:To investigate the influence of 17β-estradiol(17β-E2)in the proliferation and differentiation capabilities of primary cultured hippocampal neural stem cells(NSCs),and to clarify its potential mechanism.Methods:The NSCs were isolated from the hippocampal tissue of SD rats within 24 h of birth,and divided into control group and 17β-E2 group.Immunofluorescence method was used to detect the expressions of Nestin and 5-ethynyl-2'-deoxyuridine(EdU)in NSCs in two groups,and the proliferation rates of NSCs were calculated.Western blotting method was used to detect the expression level of Nestin protein.Flow cytometry was used to analyze the percentages of NSCs at different cell cycles.Immunofluorescence method was used to identify the expressions of markers for neuronsβⅢ-tubulin and astrocyte marker glial fibrillary acidic protein(GFAP)in the NSCs after differentiation,and the relative ratio of neurons to astrocytes was calculated.Western blotting method was used to detect the expression levels ofβⅢ-tubulin and GFAP proteins as well as phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),glycogen synthase kinase-3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),and beta-catenin(β-catenin)proteins related to PI3K/Akt/GSK-3βpathway.Results:Immunofluorescence assay revealed positive Nestin expression in NSCs in two groups;compared with control group,the proliferation rate of NSCs in 17β-E2 group was increased(P<0.01).Compared with control group,the expression level of Nestin protein in the NSCs in 17β-E2 group was increased(P<0.05),and the percentage of NSCs in S phase was increased(P<0.01).Compared with control group,the relative ratio of neurons to astrocytes in 17β-E2 group was significantly increased(P<0.05).After differentiation,compared with control group,the expression level ofβⅢ-tubulin protein in the NSCs in 17β-E2 group was increased(P<0.05),and the expression level of GFAP protein was decreased(P<0.01),while the expression levels of Akt,p-Akt,β-catenin,a

关 键 词：17Β-雌二醇 神经干细胞 细胞增殖 细胞分化 磷脂酰肌醇3-激酶 蛋白激酶B 糖原合成酶激酶3Β 

分 类 号：R741[医药卫生—神经病学与精神病学]